During year 03 of this grant I will investigate the role of calcium in the regulation of parotid gland physiology by automonic agonists. These studies will be threefold. First, the effects of agonists on 45Ca uptake and efflux will be studied in the presence and absence of agents, such as tetracaine, Co2 ion, erapamil, and neomycin, which inhibit Ca2 ion binding or flux across the cell membrane. The alterations in Ca flux will be correlated with the concurrent changes in K ion efflux and amylase release. I want to find out if certain responses can still take place when a flux is blocked. Previous work was determined that buffer Ca2 ion is required for alpha-adrenergic and cholinergic agonist action on K ion efflux. I will attempt to determine if the same is true for the changes in Ca permeability induced alpha-adrenergic and cholinergic agonists. Second, following the characterization of the regulation of Ca movements in the rat parotid I will attempt to obtain effects of agonists with sub-cellular organelles such as plasma membrane vesicles, mitochondria and microsomes derived from the parotidovinar cells. These experiments will involve either isolating the organelles from cell preparations pretreated with the agonist in question or by adding the agonists to the isolated sub-cellular organelles directly. The metabolism of Ca in these fractions will be monitored with 45Ca. Third, I will test the hypothesis that a calcium regulatory protein (CRP) mediates the action of Ca in the parotid. I have recently succeeded in purifying the rat parotid CRP to homogeniety in two steps (heat treatment coupled with isoelectric focussing). The action of purified CRP and Ca2 ion on parotid adenylate cyclase and phosphodiesterase activity will be characterized.